Bioz Ratings For Life-Science Research

Full Text

PDF

Nucleic acids research

Lipid kinase PIP5K1A regulates let-7 microRNA biogenesis through interacting with nuclear export protein XPO5.

PDF

Article Details

Authors

Chun Li, Bohyung Yoon, Giovanni Stefani, Frank J Slack

Journal

Nucleic acids research

PM Id

37655623

DOI

10.1093/nar/gkad709

Table of Contents

Abstract

icroRNAs (miRNAs) are small non-coding RNAs rst discovered in Caenorhabditis elegans . The letmiRNA is highly conserved in sequence, bioenesis and function from C. elegans to humans. uring miRNA biogenesis, XPO5-mediated nuclear xport of pre-miRNAs is a rate-limiting step and, herefore, might be critical for the quantitative conrol of miRNA levels, yet little is known about how his is regulated. Here we show a novel role for ipid kinase PPK-1 / PIP5K1A (phosphatidylinositol-4hosphate 5-kinase) in regulating miRNA levels. We ound that C. elegans PPK-1 functions in the lin8 / let-7 heter ochr onic pathwa y, which regulates the trict developmental timing of seam cells. In C. elgans and human cells, PPK-1 / PIP5K1A regulates et-7 miRNA levels. We investigated the mechanism urther in human cells and show that PIP5K1A ineracts with nuclear export protein XPO5 in the nuleus to regulate mature miRNA levels by blocking he binding of XPO5 to prelet-7 miRNA. Furthermore, e demonstrate that this r ole f or PIP5K1A is kinasendependent. Our stud y uncover s the novel finding f a direct connection between PIP5K1A and miRNA iogenesis. Given that miRNAs are implicated in muliple diseases, including cancer, this new finding ight lead to a novel therapeutic opportunity. p G f i l i t

Published online 1 September 2023 Nucleic Acids Research, 2023, Vol. 51, No. 18 9849–9862 https://doi.org/10.1093/nar/gkad709 Lipid kinase PIP5K1A regulates let-7 microRNA biogenesis through interacting with nuclear export protein XPO5 Chun Li * , Bohyung Yoon, Giovanni Stefani and Frank J. Slack * Harvard Medical School Initiative for RNA Medicine, Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA Received July 27, 2022; Revised August 03, 2023; Editorial Decision August 07, 2023; Accepted August 15, 2023 A M fi 7 g D e t t t l p f 2 s e l f t c t w i o b t m G I M n v 7 m p g i s * C P B G © T p D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 20 BSTRACT icroRNAs (miRNAs) are small non-coding RNAs rst discovered in Caenorhabditis elegans . The letmiRNA is highly conserved in sequence, bioenesis and function from C. elegans to humans. uring miRNA biogenesis, XPO5-mediated nuclear xport of pre-miRNAs is a rate-limiting step and, herefore, might be critical for the quantitative conrol of miRNA levels, yet little is known about how his is regulated. Here we show a novel role for ipid kinase PPK-1 / PIP5K1A (phosphatidylinositol-4hosphate 5-kinase) in regulating miRNA levels. We ound that C. elegans PPK-1 functions in the lin8 / let-7 heter ochr onic pathwa y, which regulates the trict developmental timing of seam cells. In C. elgans and human cells, PPK-1 / PIP5K1A regulates et-7 miRNA levels. We investigated the mechanism urther in human cells and show that PIP5K1A ineracts with nuclear export protein XPO5 in the nuleus to regulate mature miRNA levels by blocking he binding of XPO5 to pre- let-7 miRNA. Furthermore, e demonstrate that this r ole f or PIP5K1A is kinasendependent. Our stud y uncover s the novel finding f a direct connection between PIP5K1A and miRNA iogenesis. Given that miRNAs are implicated in muliple diseases, including cancer, this new finding ight lead to a novel therapeutic opportunity. p G f i l i t To whom correspondence should be addressed. Tel: +1 617 735 2632; Email: fs orrespondence may also be addressed to Chun Li. Email: cli6@bidmc.harvard r esent addr esses: ohyung Yoon, i-SENS, Inc. 43 Banpo-daero 28-gil, Seocho-gu, Seoul 06646, South iovanni Stefani, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. C The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Ac his is an Open Access article distributed under the terms of the Creati v e Common ermits unrestricted reuse, distribution, and reproduction in any medium, provided th 24 RAPHICAL ABSTRACT NTRODUCTION icroRN As (miRN As) such as lin-4 and let-7 are small on-coding RN As, w hich wer e first discover ed through deelopmental studies in Caenorhabditis elegans ( 1 , 2 ). The letmiRNA is highly conserved among species including huans ( 2 , 3 ). In the canonical miRN A bio genesis pathway, rimary miRNAs (pri-miRNAs) are transcribed from their enetic locus by RN A pol ymerase II (Pol II) and processed nto precursor miRNAs (pre-miRNAs) by the micr opr ocesor complex (consisting of Drosha and DGCR8) and exorted to the cytoplasm via an exportin 5 (XPO5) / RanTP complex ( 4–6 ). In the cytoplasm, the pre-miRNA is urther processed by the RNase III endonuclease Dicer nto small RNAs of a pproximatel y 21 nucleotides and then oaded into Argonaute proteins in the RNA-induced silencng complex (RISC) ( 4 , 5 ). Finall y, miRN As interact with he 3 ′ untranslated region (3 ′ UTR) of target mRNAs to in- lack@bidmc.harvard.edu .edu Korea. ids Research. s Attribution License (http: // creati v ecommons.org / licenses / by / 4.0 / ), which e original work is properly cited. D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 2024 duce mRNA degradation and translational r epr ession ( 4 , 5 ). During miRN A bio genesis, XPO5-mediated nuclear export of pre-miRNAs is a rate-limiting step for miRN A bio genesis ( 7 , 8 ) and, ther efor e, might be critical for the quantitati v e control of global miRNA le v els. The C. elegans heter ochr onic gene pa thway regula tes the timing of de v elopmental e v ents during post-embryonic development ( 9 ). This pathway consists of genes that encode RNA-binding proteins, such as LIN-28 and LIN-41, microRNAs, such as lin-4 and let-7 and transcription factors, such as lin-14, hbl-1 and lin-29 ( 1 , 2 , 10 , 11 ). C. elegans seam cells are lateral epidermal cells that divide during each larval stage with a stem-cell like pattern of cell fate and cell division ( 9 , 11 ). At each larval stage, one daughter of the cell division dif ferentia tes while the other daughter retains the ability to divide again. Around the beginning of the adult stage seam cells exit the cell cycle, fuse together and secrete a cuticular structure called alae ( 11 ). Mutations in heter ochr onic genes lead to temporal alterations to stagespecific patterns of cellular fate ( 11 ). For example, in precocious mutants, cells inappropriately express later cell fates during earl y stages, w hile in retarded m utants, cells reiterate earlier stage fates in place of later cell fates ( 12 ). These types of defects in de v elopmental timing result in easily scorable phenotypes, for example by using the cell fusion marker ajm-1::gfp (apical junction marker), adult cell fate marker col-19::gfp (adult specific marker) and cell terminal differentiation markers such as alae formation. Two major components of the heter ochr onic gene pathway, LIN-28 and let7 miRNA, are e volutionarily conserv ed in animals where they have been shown to regulate each other’s expression and have pivotal roles in pluripotency and dif ferentia tion ( 3 , 13 , 14 ). Howe v er, the mechanisms of action and downstream effectors of the lin-28 / let-7 pathway are poorly understood. Ther efor e, the identity of additional genes in the lin-28 / let-7 pathway in C. elegans will be important to better understand seam cell de v elopment and miRN A bio genesis. Phospha tid ylinositol 4-phospha te 5-kinase (PIP5K1A), is an enzyme that catalyzes phosphatidylinositol-4phosphate (PI4P) to phosphatidylinositol-4,5-bisphosphate (PIP2) ( 15 ). Previous studies showed that PIP2 directly interacts with RN A pol ymerase II, and also binds with nuclear protein histone H1 to counteract the histone H1-mediated r epr ession of basal transcription by RNA polymerase II ( 16 , 17 ). Larsson et al. showed human phospha tid ylinositol 4-phospha te 5-kinase (PIP5K1A) has an important role in the PI3K / AKT signaling pathway that pr omotes pr ostate cancer cell pr oliferation and survival ( 18 ). C. elegans expresses only one PIP5K1A homologue, named PPK-1. Depletion of PPK-1 results in a defect in ovulation, reduced sterility and gonadal-sheath contractility, which is associated with Ins ( 1 , 4 , 5 ) P3 signalling ( 19 ). In this study, we found that C. elegans PPK-1 regulates miRNA le v els and functions in the lin-28 / let-7 heter ochr onic pathway. Interestingly, we also found PIP5K1A (the ortholog of PPK-1) regulates miRNA le v els through interactions with nuclear export protein XPO5 in the nucleus, independent of its kinase activity. Furthermore, we demonstra ted tha t PIP5K1A blocks the binding of XPO5 to premiRNA. Ther efor e, this study describes the novel finding of a direct connection between the lipid kinase PIP5K1A and miRN A bio genesis. MATERIALS AND METHODS C . eleg ans strains The following C. elegans strains were used: N2 Bristol (wild-type)(CGC), lin-28(n719)(CGC), let-7(n2853) ( 2 ), syIs78[ajm-1::gfp + unc-119(+)] ( 20 ), maIs105[col19::gfp] ( 21 , 22 ), wIs79 [ajm-1::gfp; scm-1::gfp] ( 20 ), lin-29(n546); maIs105[col-19::gfp] ( 23 ), let-7(n2853); maIs105[col-19::gfp] ( 24 ), lin-29(n546); wIs79[ajm-1::gfp; scm-1::gfp] ( 20 ) and let-7(n2853); wIs79[ajm-1::gfp; scm-1::gfp] ( 20 ). Transgenic animals were obtained as described. Pscm::mCherry::ppk-1 (25 ng / ul) was used in PPK-1 ov ere xpression (OE) strain [Pscm::mCherry::ppk1 + Pmyo-2::dsredm]. Chromosomal integration of extr achromosomal tr ansgenes was performed using the UV integration method ( 25 ). The integrated animals were backcrossed with wild-type animals three times to clear background mutations. The Pscm::mCherry::ppk-1 plasmid was kindly provided by Dr Hitoshi Sawa. C. elegans strains were maintained a t 20 ◦C on nema tode growth medium (NGM) plates overlaid with Esc heric hia coli OP50 strain, except for let-7(n2853) which was maintained at 15 ◦C. RNA interference (RNAi) RNAi experiments were performed at 20 ◦C using E. coli HT115 bacteria expressing RNAi constructs from the Ahringer libr ary ( 26 ). Tr ansformed bacteria were over laid on a nematode growth medium (NGM) plate containing 1mM IPTG and 50ug / ml Carbenicillin. Bacteria containing L4440 vector was used as a control. Microscopic analysis L4 animals (P0) were placed on ppk-1 (RNAi) plates and the F1 progeny was scored for seam cell number ( scm-1::gfp) , seam cell fusion ( ajm-1;;gfp ), col-19::gfp expression and alae formation. scm-1::gfp, ajm-1::gfp , col-19::gfp expression and alae formation were observed using an upright Zeiss Axioplan microscope under 40x or 63x magnification. Animals were immobilized using 1mM levamisole on 2% agarose pads. De v elopmental stage was assessed by vulval and gonad de v elopment using DIC microscopy. Confocal images were acquired with a Carl Zeiss LSM 880 microscope using the Zen black software version SP2.3. The tile scan and z-stack images were acquired with a PlanApochromat 63x / NA 1.4 objecti v e lens with 10–16 images per condition. Cross-sections of a 3D volume reconstruction were generated using the Imaris image analysis software (Bitplane). Plasmids pcDNA3.1-HA-XPO5 plasmid was generated by cloning the xpo5 cDNA, amplified from HEK293 cells cDNA, into the pcDN A3.1-HA vector. pcDN A3.1-PIP5K1AFLAG plasmid was generated by cloning the pip5k1a c p t 3 M m D R A s s i a l a i w s i p p s t t e G p m ( m e e p o m t ( p m w t a s t c 4 c R s o G w l t l m C o f s • • • • • • • • • • • • • • • • • • • • • • b h s c 7 ( M G H L H U 0 ( h I N T o n l t a m the membranes were further UV-cross-linked and dried at D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 2024 DNA, amplified from HEK293 cells cDNA, into the cDNA3.1-FLAG vector. The kinase-dead PIP5K1A muant ( PIP5K1A -D309N) was generated by replacing Asp09 with Asn, which was performed by a Q5 Site-Directed utagenesis Kit (New England Biolabs). HA-KAP1 plasid was kindly provided by Dr Dipanjan Cho w dhury from ana-Farber Cancer Institute / Harvard Medical School. NA isolation and qRT–PCR nimals were bleached (5% 5 N NaOH, 10% bleach in M9 olution) starved in M9 (without E. coli ) overnight to get ynchronized L1s, which were put on plates for the experments. qRT-PCR (Figures 1 B and 2A– H) was performed t the peak time point of lin-42 mRNA expression levels, as in-42 mRNA is dynamically expressed during development nd peaks during the L4 stage ( 27 ). Total RNA was collected in TRIzol (Invitrogen) and solated using PureLink ™ RNA Mini Kit (Invitrogen) or ith Dir ect-zol Minipr ep Plus spin columns (Zymo Reearch) according to the manufacturer’s protocol, includng the on-column DNase I treatment. cDNA synthesis was erformed using High-Capacity RNA-to-cDNA ™ Kit (Aplied Biosystems) or High-Capacity cDNA Re v erse Trancription Kit (Applied Biosystems). Total RNA concentraion was measured using the NanoDrop Spectrophotomeer (ND-1000 Spectrophotometer). mRNA expression levls were determined by quantitati v e RT-PCR using SYBR reen (Applied Biosystems) according to manufacturer rotocols (Roche). MiRNA expression levels were deterined by RT-qPCR using TaqMan MicroRNA Assays A pplied Biosystems). MiRN A le v els in animals were noralized to U18 snoRNA, while mRNA or pri-miRNA levls were normalized with pmp-3 or actin ( act-1) mRNA levls and 5.8S rRNA served as control for normalization of re-miRNA le v el. For mammalian RNA samples, miRNA r pre-miRNA and mRNA or pri-miRNA le v els were noralized to U6 snRNA and GAPDH mRNA, respecti v ely. Nuclear and cytoplasmic RNAs were isolated following he protocol by Gagnon et al ( 28 ) and Choudhury et al 29 ). Briefly, RKO cells were l ysed in ice-cold HLB (Hyotonic lysis buffer: 10 mM Tris, pH 7.5, 10 mM NaCl, 3 M MgCl 2 , 0.3% NP-40 and 10% glycerol), supplemented ith 100 U of Ribolock RNase inhibitor and incubated he mixture on ice for 10 min followed by centrifugation t 1000 g at 4 ◦C. for 3 min. We carefully transferred the upernatant (cytoplasmic fraction) to a new tube and kept he pellet on ice. The nuclear pellet w as w ashed with iceold HLB buffer three times and centrifuged at 300 g at ◦C. for 2 min. TRIzol was then added to both nuclear and ytoplasmic fractions and proceeded for RNA extraction. T-qPCR was performed using TaqMan MicroRNA asays (Applied Biosystems), as described earlier. The purity f cytoplasmic and nuclear fractions was determined using APDH mRNA and MALAT1 , respecti v ely. Experiments ere done in triplicate. For validation of the percent of preet-7a-1 / pri- let-7a-1 in the cytoplasmic-nuclear fractionaion experiment, first we calculated the C / N ratio of preet-7a-1 or pri- let-7a-1 by using the 2 − Ct [2 −(Cyt(ct)- Nuc(ct)) ] ethod ( Ct method). From this result, we calculated the / N ratio of pre- let-7a-1 / pri- let-7a-1 , to get the percent f pre- let-7a-1 / pri- let-7a-1 in the cytoplasmic or nuclear ractions. Primers (SYBR Green-based qPCR) for used in this tudy are: act-1 -f: 5 ′ -ACGCCAACACTGTT CTTT CC-3 ′ act-1 -r: 5 ′ -GATGAT CTTGAT CTTCAT GGTT GA-3 ′ pmp-3 -f: 5 ′ -GTT CCCGTGTT CAT CACT CAT-3 ′ pmp-3 -r: 5 ′ -A CA CCGTCGA GAA GCTGTA G-3 ′ ppk-1 -f: 5 ′ -AAAGCT CGGACAT CGACGAA-3 ′ ppk-1 -r: 5 ′ -GA GACGCCA GACTT CCTAT CG-3 ′ lin-28 -f: 5 ′ -GCAA GGATTTCGGA GTCTTGATGAA GG-3 ′ lin-28 -r: 5 ′ -GCAAACTTTCCA CATCTGAAGCAA C GTA-3 ′ lin-41 -f: 5 ′ - GCTTCA GCA GTT GAT GGCTAC-3 ′ lin-41 -r: 5 ′ - CAT CT CCACTT CCAACTGAT CC-3 ′ hbl-1 -f: 5 ′ -CT CGT CTAGTGACCCATT CT-3 ′ hbl-1-r: 5 ′ -A CGCCCGAA CA TTGA TAAG-3 prime; Ce.5.8S rRNA-f: 5 ′ -CTAGCTTCAGCGATGGATC GG-3 ′ Ce.5.8S rRNA-r: 5 ′ -CAACCCTGAACCAGACGTA CC-3 ′ GAPDH -f: 5 ′ -TGCA CCA CCAA CTGCTTAGC-3 ′ GAPDH -r: 5 ′ -GGCAT GGACT GT GGTCAT GAG-3 ′ PIP5K1A -f: 5 ′ -CT CCGGGCCGT CGT CTT CG-3 ′ PIP5K1A -r: 5 ′ -GCATAA GGCACCTCA GATGC-3 ′ U6-f: 5 ′ -CT CGCTT CGGCAGCACA-3 ′ U6-r: 5 ′ -AA CGCTTCA CGAATTTGCGT-3 ′ XPO5 -f: 5 ′ -TGGCCACA GA GGTCACCC-3 ′ XPO5 -r: 5 ′ -GGGCGCAGTGCCTCGTAT-3 ′ TaqMan probes (Thermo Fisher Scientific) are shown elow. U18 (Assay ID: 001764), cel-lin-4 (Assay ID: 000258), sa-let-7a (Assay ID: 000377), cel-miR-75-3p (Asay ID: 000288), cel-miR-77-3p (Assay ID: 000230), el-miR-237–5p (Assay ID: 462482 mat), pri-hsa-leta-1miRN A (Hs03302533 pri), pri-hsa-let-7b miRN A Hs03302548 pri), Hsa-pre-let-7a-1 (Custom Taq- an Gene Expression assa y, Assa y ID: AP4729Y), APDH (Assay ID: Hs02786624 g1), -actin (Assay ID: s01060665 g1), HMGA2 (Assay ID: Hs04397751 m1), IN28A (Assay ID: Hs04189307 g1), MYC (Assay ID: s04189307 g1), MALAT1 (Assay ID: Hs00273907 s1), 6 snRNA (Assay ID: 001973), hsa-let-7b (Assay ID: 00378), has-let-7c (Assay ID: 000379), hsa-miR-125a-5p Assay ID: 002198), hsa-miR-125b-5p (Assay ID: 000449), sa-miR-886-5p (Assay ID: 002193), hsa-miR-122(Assay D:002245). orthern blotting otal RNA (20–40 ug) was extracted from nematodes r cells using TRIzol reagent (Invitrogen, USA) and orthern blots performed using biotin-labeled probes fol- owing the protocol by Gagnon et al. ( 28 ). Briefly, toal RNA samples were run on 15% PAGE–urea gels nd transferred to Hybond-N+ positi v ely charged nylon embranes (GE healthcar e, USA) by electrophor esis. Next, D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 2024 60 ◦C for 1 h to improve binding. Before hybridization, the membranes wer e pr e-hybridized for at least 1 h at 40 ◦C in pre-hybridization buffer (#AM8677, Thermo Scientific). Next, hybridiza tion buf fer containing 50 pmol / ml biotin labeled single-stranded DNA oligonucleotide (See below) was added and the membranes were hybridized for overnight at 40 ◦C with gentle shaking and subsequently rinsed with low stringency wash buffer (#AM8673, Thermo Scientific) 3 times and High stringency wash buffer 2 times (#AM8674, Thermo Scientific) at room temperature. The biotin-labeled probes were detected using a Chemiluminescent Nucleic Acid Detection Module Kit (#89880, Thermo Scientific) following the manufacturer’s instructions. The bands were quantified using the ImageJ software. • Hsa - let-7a : 5 ′ -AACT AT ACAACCTACT ACCTCA -3 ′ / Bio / • Ce.5.8S: 5 ′ -GAACCAGA CGTA CCAA CTGGAGGCC C -3 ′ / Bio / • Hsa.5.8S: 5 ′ -TCCTGCAA TTCACA TTAA TTCTCGC AG -3 ′ / Bio / Immunoprecipitation 48h after transfection with the indicated plasmids, HEK293T cells were harvested and lysed with IP lysis buffer (Thermo Scientific ™) supplemented with Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) and incubated on ice for 20 min. Human cells samples were centrifuged at 15 000 rpm at 4 ◦C for 10 min. 5% of the supernatant was separated as input and the rest of the supernatant was incubated with anti-FLAG antibody (#F1804, Millipore sigma), anti-mCherry (#43590, CST) antibody, anti-HA magnetic beads (Thermo Scientific) and anti-PIP5K1A (pre-conjugated with protein A magnetic beads) overnight with constant rotation at 4 ◦C. Later, beads were washed using wash buffer and the bound protein was eluted with 4 × Laemmli Sample Buffer (Bio-Rad) by boiling for 5 min and further analyzed by western blot. Western blotting Animals were lysed by boiling in SDS sample buffer while cells were harvested and lysed in RIPA lysis buffer (Thermo Scientific ™) supplemented with protease inhibitor cocktail (Thermo Scientific). Total protein concentration was determined using Pierce BCA protein assay kit (Thermo Scientific). Proteins resolved by SDS page were transferred to PVDF membr ane. Membr anes were blocked with 5% milk protein in 1 × TBST and incubated with primary antibodies overnight. Membranes were washed three times with 1 × TBST and probed with a HRP-conjugated secondary antibody for 1 h followed by three additional washes. Specific antibody binding onto the membranes was detected using the SuperSignal ™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). The following antibodies were used: V5-tag Polyclonal antibody (#14440-1-AP, Thermo scientific), Monoclonal ANTI-FLAG ® M2 antibody (#F1804, Millipore sigma), DYKDDDDK Tag (D6W5B) Rabbit mAb (#14793, CST), GAPDH (14C10) Rabbit mAb (#2118, Cell signaling), Anti-HA antibody (#H3663, Millipore sigma), B-Actin antibody (#sc-47778, Santa Cruz Biotechnology), XPO5 Polyclonal Antibody (#PA593224, onl y reco gnizes the C-terminus of XPO5, URL: https://www.thermofisher.com/antibody/product/XPO5Antibody- Polyclonal/PA5- 93224 ), PIP5K1A Polyclonal Antibody (#15713-1-AP, Thermo scientific), Anti-rabbit IgG HRP-linked Antibody (#7074, Cell signaling), Antimouse IgG, HRP-linked Antibody (#7076, Cell signaling). Cell lines and transfection HEK293T (obtained from the American Type Culture Collection (ATCC)) and RKO (Provided by Dr Kevin Haigis from Dana-Farber Cancer Institute of Harvard Medical School) cell lines wer e cultur ed in DMEM (high glucose) (Gibco, Cat #11995-065) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin–streptomycin. Each cell line was maintained in a 5% CO 2 atmosphere at 37 ◦C. To generate stable knockdown cell lines, PIP5K1A ShRNA (TRCN0000231477, Millipore Sigma) and control ShRNA (SHC016, Millipore Sigma) constructs were first transfected into HEK293T cells with the VSVG envelope vector and psPAX2 packaging vector using TransIT-Lenti reagent according to manufacturer’s protocol (MirusBio). After 48 h, media was collected, centrifuged, and filtered through a 0.45 m nitrocellulose filter. The virus was then added to RKO cells, and stably transduced cells were selected with puromycin and were maintained in 1.0 g / ml puromycin media. To generate PIP5K1A -WT OE or PIP5K1A -D309N OE cells, pcDNA3.1-PIP5K1A-FLAG or pcDNA3.1-PIP5K1A- D309N-FLAG were transfected to RKO cells respecti v ely , by using T ransIT-X2 reagent (Mirus Bio, Madison, WI, USA) following the manufacturer’s protocol. The plasmids and small interfering RNAs (siRNAs) were transfected to HEK293T or RKO cell lines using the TransIT-X2 reagent (Mirus Bio, Madison, WI, USA) following the manufacturer’s protocol. Silencer select siRNAs wer e pur chased from Santa Cruz Biotechnology: XPO5 siRNA (sc-45569) and Control siRNA (sc-37007). Immunofluorescent staining Samples wer e fix ed in 4% PFA for 10 min in phosphatebuffered saline (PBS) and washed three times for 5 min in PBS at room temperature, then incubated with 3% BSA in 0.3% Triton X-100 in PBS for 30min at room temperature. Samples were incubated with primary antibodies overnight a t 4 ◦C , and washed three times. Later they were incubated with secondary antibodies for 1 hour at room temperature, washed and mounted with ProLong ™ Gold Antifade Mountant with DAPI ((#P36931, Thermo Fisher). Primary antibodies: PIP5K1A Polyclonal Antibody (# 15713-1-AP, Thermo Fisher scientific), XPO5 purified MaxPab mouse polyclonal antibody (B01P) (# H00057510-B01P, Abnova), Fluorescein conjugated Anti-PI ( 4 , 5 ) P2 IgM (#ZG045, Echelon Biosciences), DYKDDDDK Tag (D6W5B) Rabbit mAb (Alexa Fluor ® 594 Conjugate) (#20861, Cell signaling). Secondary antibodies: Goat anti-Rabbit IgG Secondary Antibody Alexa Fluor 488 (# A27034, Thermo Fisher scientific), Goat anti-Mouse IgG Secondary Antibody Alexa Fluor 568 (# A-11004, Thermo Fisher scientific). Confocal images were acquired with a Carl Zeiss L W a i t p R R t e i s P ( a 1 s t b a w p c a U i ( n R F c t H m m t X a T w 1 P b ( p t G R p s S A i d a * s R p C w ( i 2 h l o l a s l i t t P 1 7 s s w t s s y l d ( m c f e 1 n 1 1 e 1 m a s m L a 1 a u m ( w T p s D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 2024 SM 880 upright confocal microscope, as described above. e define the C / N ratio of subcellular localization of XPO5 s the ratio of cytoplasmic to nuclear immunofluorescence ntensity of XPO5, assessed by the mean fluorescence inensity in the cytoplasmic and nucleus volumes, which was erformed by ImageJ software. NA immunoprecipitation assay (RIP) KO control and PIP5K1A knockdown cells were ransfected with plasmids expressing HA-XPO5. Fortyight hours after transfection, the plates were rinsed twice n ice-cold PBS, cross-linked at 254 nm (120 mj / cm 2 ), craped and lysed in IP lysis buffer supplemented with rotease Inhibitor Cocktail and Ribolock RNase Inhibitor 40 U / l; Thermo Fisher Scientific) on ice for 20 min nd then centrifuged at 15 000 rpm speed at 4 ◦C for 0 min. After centrifugation, 10% of the supernatant was eparated as the input for qRT-PCR analysis. The rest of he supernatants were incubated with anti-HA magnetic eads or control magnetic beads with constant rotation t 4 ◦C for overnight. The beads were washed 4 times with ash buffer. After washing, the beads were treated with roteinase K, and RNA was purified as described above. DNA synthesis and PCR was performed as described bove. After qRT-PCR analysis, the levels of pre- let-7 and 6 in the IP samples were divided by the levels of them n the input calculated by a relati v e enrichment method, 2 –(IP(ct)) – (input(ct) . Fold enrichment of this ratio for HA IP ormalized to the negati v e control IgG IP was determined. NA pull-down assay or GTP loading, 100uM GTP gamma S (#ab146662, Abam) was incubated with Ran (#PRO-841, Prospec) proein and 20mM EDTA in the loading buffer (50 mM EPES (pH 7.3), 200 mM NaCl, 5 mM MgCl 2 , 5 mM - ercaptoethanol) at 30 ◦C for 30 min. After loading, 60 M MgCl 2 was added and the reaction incubated on ice o stop the loading. RKO cells were transfected with HAPO5, lysed and XPO5 immunoprecipitated with anti-HA ntibody and then eluted by IgG elution buffer (#21028, hermo Fisher). Eluted HA-XPO5 protein was incubated ith the above RAN-GTP, 3 ′ biotin labeled pre- let-7a(Purchased from GenScript) with or without of GSTIP5K1A (#ab261922, Abcam) at 4 ◦C f or 4h, f ollowed y incubation with Dynabeads MyOne Streptavidin C1 #65001, Thermo Fisher) for another 1 h a t 4 ◦C . La ter, the ull-downed proteins were washed using wash buffer and hen subjected to western blotting by anti-HA and antiST antibodies (#2625, Cell Signaling). For detecting the N A pull down efficientl y, Streptavidin bead pull-downed re- let-7a-1 was run on 15% PAGE–urea gels and then tained by GreenGlo Dye (#CA3600, Fisher Scientific). tatistical analysis ll experiments were performed with at least three biologcal replica tes. Sta tistical analysis was performed by Stuent’s two- tailed t test in GraphPad Prism 9. Data points r e pr esented as the mean ± SEM. P -values are: * P < 0.05, * P < 0.01, *** P < 0.001, **** P < 0.0001 and NS = not ignificant. ESULTS pk-1 is a heter ochr onic gene . elegans heter ochr onic genes in the lin-28 / let-7 pathay regulate the strict de v elopment timing of seam cells 2 , 11 ). We pr eviously r eported the use of CLIP-Seq to dentify 2000 mRNAs interacting directly with the LIN8 RNA binding protein ( 30 ), some of which were known eter ochr onic genes. To enrich for additional genes in the in-28 / let-7 pathway, we determined the overlap among ur set of LIN-28 CLIP hits ( 30 ), the set of 201 known et-7 suppressors ( 31 ), 213 known let-7 enhancers ( 32 ), nd an analysis of lin-28 dependent genes from our curory RNA-seq data (Supplementary Table S1) from staged ate larval stage 1 (L1) WT and lin-28 ( n719 ) mutant anmals. We found 16 candida te genes tha t are a t the inersection of these groups (Supplementary Figure S1). Of hese genes, we focused on ppk-1 , an ortholog of human IP5K1A (phospha tid ylinositol-4-phospha te 5-kinase type alpha). While ppk-1 was identified in the 213 known letenhancers ( 32 ) group, in that study, it did not show a trict positi v e relationship with let-7 , as the vulval burst core was only 1.1 ( 32 ). PPK-1 / PIP5K1A is an enzyme hich produces phospha tid ylinositol-4-phospha te (PI4P) o phospha tid ylinositol-4,5-bisphospha te (PIP2), a lipid econd messenger tha t regula tes se v eral cellular processes, uch as signal transduction and vesicle trafficking ( 33 ). Since PPK-1 was identified from LIN-28 CLIP-seq analsis, we tested if LIN-28 potentially regulates ppk-1 mRNA e v els. In lin-28 ( n719 ) mutants, ppk-1 mRNA le v els were ecr eased compar ed to wild type animals at the L2 stage Figure 1 A) (Supplementary Table S1). Surprisingly, lin-28 RNA le v els also decreased in ppk-1 RNAi compared to ontrol RNAi animals (Figure 1 B), suggesting that they unction on each other in a feed forward loop. To determine whether ppk-1 is a heter ochr onic gene, we xamined the well-characterized cell fusion marker ajm::gfp (Apical junction marker), alae formation (cell termial dif ferentia tion marker) and adult cell fate marker col9::gfp (adult specific marker) in temporally-staged ppk(RNAi) and control RNAi animals (the ppk-1 null mutant xhibits lethality ( 34 )). We confirmed efficient RNAi of ppkmRNA by qRT-PCR (Figure 1 B). In control RNAi anials, seam cell fusion - as detected by ajm-1::gfp - occurs t the middle (mid) L4 sta ge, b ut is not observed at early tages (Figure 1 C–E). Howe v er, 15% of ppk-1 (RNAi) anials showed the ajm-1::gfp fusion phenotype at the mid3 stage, 44.4% of them at late-L3 stage and 42.9% of them t early-L4 stage (Figure 1 C–E). Similarly, 36.4% of ppk(RNAi) animals showed precocious col-19::gfp expression t the L4 sta ge, b ut control RNAi animals did not (Figr e 1 F). Furthermor e, we also found that ppk-1 (RNAi) anials showed precocious alae forma tion a t the early L4 stage 14.3%) and mid-L4 stage (45%) compared to the wild type hich onl y showed this a t the la te L4 stage (Figure 1 G, H). hese results indicate that depletion of ppk-1 causes a weak recocious heter ochr onic phenotype. Furthermore, as preented in Figure 1 I, ppk-1 (RNAi) animals showed a reduced Figure 2. PPK-1 regulates miRNA expression and functions in lin-28 / let-7 heter ochr onic pathway. ( A, E ) Northern blot analyses of mature let-7 expression from the indicated animals (at the peak time point of lin-42 le v els). 5.8S rRNA was used as a loading control. All experiments were performed with three biolo gical replicates. ( B , F ) Quantitation of let-7 / 5.8S le v els from northern b lot anal yses using ImageJ. ( C, G ) qRT-PCR anal ysis of mature let-7, lin-41 and hbl-1 le v els from the indicated animals (at the peak time point of lin-42 le v els). miRNA le v els were normalized to U18 snoRNA. lin-41 and hbl-1 mRNA le v els were normalized to act-1 mRNA. ( D ) qRT-PCR analysis of ppk-1 mRNA le v els in the wild type and PPK-1 OE animals. act-1 mRNA was used as an endo genous control. (H ) qRT-PCR anal yses of mature lin-4 , miR-75, miR-77 and miR-237 le v els in control RNAi and ppk-1 (RNAi) animals. U18 snoRNA was used as an endogenous control. All experiments were performed with at least three biological replicates. All data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001 and NS: not significant. s R p p P 2 O l o w f i d t p F s t n W 1 e s o w 1 W i ( i O c v p m c t t D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 2024 eam cell number from L2 to L4 stages compared to control NAi animals. Since these phenotypes are characteristic of recocious seam cell patterning, these results suggest that pk-1 is a new heter ochr onic gene. PK-1 regulates miRNA levels and functions in the lin8 / let-7 heter ochr onic pathwa y ne feature of precocious heter ochr onic mutants such as in-28 is altered expression of miRNAs involved in de v elpmental timing, such as let-7 ( 35 ). In order to determine hether PPK-1 regulates let-7 miRNA expression, we perormed qRT-PCR experiments over developmental time usng lin-42 as a r efer ence gene, as its expression peaks once uring each larval stage ( 27 ). ppk-1 (RNAi) animals showed he peak time point of lin-42 expression 3 hours earlier comared to control RNAi animals at 20 ◦C (Supplementary igure S2A-B). At the peak time point of lin-42 mRNA le v els, we oberved that ppk-1 (RNAi) animals showed increased maure let-7 le v els compared to control RNAi animals by orthern blotting and qRT-PCR analysis (Figure 2 A–C). e also found that mature let-7 le v els increased in ppk(RNAi) animals compared to control RNAi animals at arly stages such as m-L2 and e-L3 stage by a qRT-PCR asay, which was consistent with the precocious phenotypes f ppk-1 (RNAi) animals (Supplementary Figure S3). We examined whether over-expression (OE) of PPK-1 as sufficient to alter let-7 expression, we created PPK(OE) animals by integrating a ppk-1 multi-copy array. hen dri v en fr om its own pr omoter, the PPK-1 (OE) anmals showed sterility and embryonic lethality phenotypes not sho wn). Ho we v er, as ppk-1 was known to be expressed n seam cells from previous report ( 34 ), we created PPK-1 E integrated animals driving ppk-1 from a seam cell speific promoter. PPK-1 (OE) animals showed a 4 hour deelopmental delay compared to wild type animals (Suplementary Figure S2C, D), and showed increased ppk-1 RNA le v els (Figure 2 D). PPK-1 OE animals showed der eased matur e let-7 le v els compared to wild type animals at he peak time point of lin-42 le v els, through northern bloting and qRT-PCR analysis (Figure 2 E-G). These results D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 2024 suggest that PPK-1 negati v ely regulates the expression of mature let-7. To test if ppk-1 has a genetic interaction with let-7 in the heter ochr onic pathway, we knocked down ppk-1 in let7(n2853) mutants. We found that the let-7(n2853) mutation suppresses the precious col-19::gfp phenotypes and decreased seam cell number of ppk-1 (RNAi) animals (Figure 1 F, J). let-7 controls seam cell divisions by negati v ely regulating its targets lin-41 and hbl-1 . We found that the mRNA le v els of lin-41 significantly decreased or increased under depletion or ov ere xpression of PPK-1 compared to control animals, respecti v ely, howe v er hbl-1 mRNA le v els were not significantly changed in both conditions (Figure 2 C, G). lin-29 , encodes a zinc-finger transcription factor that is r equir ed for hypodermal seam cell terminal differentiation, and functions downstream of let-7 ( 36 ). We found that the lin-29(n546) mutation suppressed the precious phenotypes and decreased seam cell number of ppk-1 (RNAi) animals, similar to the let-7 ( n2853 ) mutation (Figure 1 F, J). These results indica te tha t PPK-1 functions upstream of let-7 / lin-41 and lin-29 . We also tested the le v els of other miRNAs, such as lin-4 , miR-75 , miR-77 and miR-237 , in ppk-1 (RN Ai) animals. Interestingl y, most of those miRN A le v els were also significantly increased in ppk-1( RNAi) animals (Figure 2 H). Taken together, these results demonstra te tha t PPK-1 regula tes miRNA le v els and acts in the lin-28 / let-7 heter ochr onic pathway. Since most of the C. elegans factors shown abov e hav e orthologs in mammalian cells, to further explore the detailed mechanism of how PPK-1 regulates mature let-7 le v els, we utilized mammalian cells going forward. PIP5K1A, the ortholog of C. elegans PPK-1 regulates miRN A e xpression As PPK-1 is an ortholog of human PIP5K1A, we determined whether PIP5K1A regulates miRNA le v els similar to C. elegans PPK-1. Firstly, we generated PIP5K1A stable knockdown (KD) in RKO colon cancer cells, a wellestablished model in miRN A bio genesis r esear ch involving pre-miRNA / XPO5 ( 37 ), by a lenti virus-based inducib le shRNA system and confirmed knockdown of PIP5K1A by qRT-PCR (Figure 3 A). Next, we investigated whether PIP5K1A regulates let-7 miRNA expression. We found that knockdo wn of PIP5K1A sho wed incr eased matur e let-7a and unchanged pre- let-7a-1 le v els compared to control cells by northern blotting and qRT-PCR assays (Figure 3 B– E). We also found that mature let-7b and let-7c le v els increased in PIP5K1A KD cells compared to control cells using a qRT-PCR assay (Figure 3 E). Furthermore, PIP5K1A KD cells showed decreased pri- let-7a-1 and pri- let-7b le v els compared to control cells (Figure 3 F). As pri- let-7c expression le v els are v ery low in these cells, we did not investigate them. We also tested the effect of ov ere xpression (OE) of PIP5K1A on let-7 le v els by northern blotting and qRT-PCR experiments. Firstly, we confirmed that PIP5K1A mRNA e xpression le v els incr eased in PIP5K1A OE cells compar ed to control cells (Figure 3 G). We found that PIP5K1A OE cells showed decreased mature let-7a and unaltered pre- let- 7a-1 le v els relati v e to wild type cells (Figure 3 H-K). Mature let-7b and let-7c le v els also decreased, as tested by qRTPCR (Figure 3 K). We also found that PIP5K1A OE cells showed increased pri- let-7a-1 and pri- let-7b le v els (Figure 3 L). Gi v en that the change in le v els of pri-miRNA and matur e miRNA ar e not concordant, these results indica te tha t PIP5K1A regulates let-7 miRNA expression at least partially at the post-transcriptional le v el. As PIP5K1A negati v ely regula tes ma ture let-7 le v els, we tested whether the knockdown of PIP5K1A effects expr ession of downstr eam targets of let-7 miRNAs, such as LIN28A , MYC , HMGA2 ( 38 ) . We found that those mRNA le v els decreased in PIP5K1A KD compared to with control cells (Figure 3 M). Furthermore, other miRNAs such as miR-122, miR-125a / miR-125b (orthologs of C. elegans lin4 miRNA ( 39 )) and miR-886 le v els increased in PIP5K1A KD cells, howe v er some not significantly (Figure 3 N). Taken together, these findings indicate that PIP5K1A negati v ely regulates mature miRNA le v els similar to its C. elegans ortholog, PPK-1. PIP5K1A physically interacts with and co-localizes with XPO5 From our results above, PIP5K1A seemed to participate in regulating miRNA processing or biogenesis. Mammalian XPO5 belongs to the importin-beta family, a Ran-GTPdependent dsRNA-binding protein that regulates the nuclear export of pre-miRNAs ( 6 , 40 ) and has a rate-limiting role in miRN A bio genesis. This led us to test whether there is a connection between PIP5K1A and XPO5. We confirmed that siRNA knockdown of XPO5 indeed results in decr eased matur e let-7a , let-7b and let-7c le v els compared to control cells using a qRT-PCR assay as previously reported ( 37 ) (Supplementary Figure S4). Firstly, we tested the simple hypothesis that PIP5K1A and XPO5 have a physical interaction. We immunoprecipitated PIP5K1A from HEK293 cell lysates with antiPIP5K1A. We found that XPO5 co-precipitated with PIP5K1A (Figure 4 A). Howe v er, immunoprecipitation of XPO5 with an anti-XPO5 antibody, did not show PIP5K1A co-precipitating with XPO5 (data not shown). As this antiXPO5 antibody only recognizes the C-terminus of XPO5 (see Materials and Methods), we created an N-terminus tagged HA-XPO5 and performed the same experiments. We found that PIP5K1A co-precipitated with XPO5 when immunoprecipitation of XPO5 was with an anti-HA antibody (Figure 4 B). We also used HA-KAP1(KRAB-associated protein) as a negati v e control to test the special interaction between PIP5K1A and XPO5. When we used HA antibody to immunoprecipitated KAP1, we did not detect PIP5K1A co-precipitating with KAP1 (Supplementary Figure S5). These results indicate that PIP5K1A physically interacts with XPO5 and seems to do so through its C-terminus. To explore this interaction in vivo , the subcellular localization of the PIP5K1A-XPO5 complex was examined by immunofluorescence. We found that PIP5K1A co-localized with XPO5 mainly in the nucleus (Figure 4 C). The presence of the colocalization signal within the nucleus was further examined by generation of a 3D cross-section recon- Figure 3. PIP5K1A regulates miRNA expression . ( A, G ) qRT-PCR analysis of PIP5K1A mRNA le v els from indicated RKO cell lines. GAPDH mRNA was used as an endogenous control. ( B, H ) Northern blot analyses of mature let-7a and pre- let-7a-1 expression from the indicated cells. 5.8S rRNA was used as a loading control. All experiments were performed with at least thr ee biological r eplica tes. (C , D, I, J) Quantita tion of let-7a / 5.8S ( C , I ) or pre- let-7a-1 / 5.8S ( D, J ) le v els using Image J from the indicated cells. ( E, K ) qRT-PCR analysis of mature let-7a, let-7b and let-7c le v els from indicated RKO cell lines. U6 snRNA was used as an endogenous control. ( F, L ) qRT-PCR analysis of pri- let-7a-1 and pri- let-7b le v els from indicated RKO cell lines. pri-miRNA le v els were normalized to GAPDH mRNA. ( M ) qRT-PCR analysis of LIN-28A , MYC and HMGA2 mRNA le v els in control and PIP5K1A KD RKO cell lines. GAPDH mRNA was used as an endogenous control. ( N ) qRT-PCR analysis of mature miR-122 , miR-125a , miR-125b and miR-886 le v els in control and PIP5K1A KD RKO cells. U6 snRNA was used as an endogenous control. All experiments wer e performed with at least thr ee biological r eplica tes. All da ta ar e r epr esented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and NS: not significant. s c c t t d ( X P m T m p k g r u m a f X t t f ( f m p i X c t c X D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 2024 tructed from a confocal z-stack, demonstrating that coloalization (yellow) of PIP5K1A (green) and XPO5 (red) ocurs in the nucleus in three dimensions (3D), as shown in he x,y, x,z and y,z planes (Figure 4 D). As the nuclear fracion of XPO5 binds with pre-miRNA in a Ran-GTP depenent manner and then exports the pre-miRNA to cytoplasm 6 , 7 ), this suggests that PIP5K1A may has a function with PO5 in nucleus. IP5K1A r egulates the pr e-miRN A / XPO5 comple x during iRNA biogenesis o address the question of how PIP5K1A functions in iRN A bio genesis, we tested whether PIP5K1A affects exression and / or function of XPO5. Because XPO5 is a nown factor that has an important role in miRNA bioenesis and PIP5K1A associates with XPO5 from the above esults, we tested the simple hypothesis that PIP5K1A reglates XPO5 le v els and that accounts for its contribution to c iRN A bio genesis. Thus, we anal yzed the transcriptional nd translational le v els of XPO5 in PIP5K1A KD cells. We ound that PIP5K1A does not affect XPO5 mRNA and PO5 protein le v els (Figure 4 E, F). This result indicates hat PIP5K1A neither regulates XPO5 transcriptionally nor ranslationally. As the C-terminal region of XPO5 is essential for the ormation of the pre-miRNA / XPO5 / Ran-GTP complex 37 , 41 , 42 ) and it is also important to interact with PIP5K1A rom our results, this led us to hypothesize that PIP5K1A ay have a role in the process of the formation of the re-miRNA / XPO5 comple x. For e xample, PIP5K1A bindng may block pre-miRNA binding to the C-terminus of PO5. If this is true, knockdown of PIP5K1A should inrease the binding ability of pre-miRNA with XPO5. To est this hypothesis, we performed an RN A imm unopreipitation (RIP) assay in RKO cells. We expressed HAPO5 in PIP5K1A KD and control cells and immunopreipitated XPO5 with the anti-HA antibody. We checked Figur e 4. PIP5K1A physicall y interacts with and localizes with XPO5. (A, B) Physical interaction between PIP5K1A and XPO5. ( A ) Endogenous PIP5K1A of HEK293 cells was immunoprecipitated and then the XPO5 protein was analyzed by immunoblotting. Normal IgG was used as a negati v e control. ( B ) HEK293 cells were transfected with HA::XPO5 and XPO5 was immunoprecipitated with anti-HA antibody. Normal IgG was used as a negati v e. ( C ) Representati v e image of confocal imaging of co-localization between PIP5K1A and XPO5 in RKO cells. DAPI was used to stain the nuclear DNA. n = 30 cells, Scale bar: 7 m. ( D ) Cross-sections of a 3D volume reconstruction. The presence of the colocalization signal was examined by generation of a 3D cross-section reconstructed from a confocal z-stack. The colocalization (yello w, arro w) of PIP5K1A (green) and XPO5 (red) in the nucleus in three dimensions (3D), as shown in the x,y, x,z, and y,z planes. ( E ) qRT-PCR analysis of XPO5 mRNA le v els in control and PIP5K1A KD RKO cells. GAPDH mRNA was used as an endogenous control. The data are represented as mean ± SEM. Individual experiments were performed in triplicate. NS: Not significant. ( F ) Western blot of XPO5 protein levels in control and PIP5K1A KD RKO cells. GAPDH mRNA was used as a loading control. D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 2024 for XPO5 protein le v els by western blot analysis (Figure 5 A). Interestingly, we found tha t XPO5-associa ted pre- let7a-1 le v els dramatically increased in cells with knockdown of PIP5K1A compared to control cells (Figure 5 B). As a negati v e control, U6 snRNA le v els were not changed (Figure 5 B). Moreover, we used 3 ′ biotin labeled pre- let-7a1 and performed an RNA pull-down assay. We expressed HA-XPO5 in RKO cells and immunoprecipitated XPO5 with the anti-HA antibody. Eluted HA-XPO5 protein was incubated with RAN, GTP and 3 ′ biotin labeled pre- let-7a1 with or without GST-PIP5K1A. The experiment showed tha t pre- let-7a-1 -associa ted HA-XPO5 le v els were significantly reduced by addition of GST-PIP5K1A, compared to that without PIP5K1A (Figure 5 C, D). We also confirmed the RNA pulldown efficiency and did not see a difference under these conditions (Supplementary Figure S6). Furthermore, we also examined the cellular localization of XPO5 under depletion or ov ere xpression of PIP5K1A Figure 5. PIP5K1A regulates the binding ability of pre-miRNA / XPO5 complex . (A, B) RNA immunoprecipitation (RIP) assay. ( A ) RIP assay was employed by using anti-HA antibody and normal IgG antibody in control and PIP5K1A KD RKO cells. HA::XPO5 was transfected in control and PIP5K1A KD RKO cells and then HA::XPO5 was immunoprecipitated with anti-HA antibody. Normal IgG was used as a negati v e control. XPO5 protein was analyzed with anti-HA antibody by immunoblotting. GAPDH was used as a loading control. ( B ) Fold enrichment of pre- let-7a-1 detected by qRT-PCR. U6 snRNA was used as a negati v e control. n = 6 independent biological replicates. ( C ) Western blot of XPO5 protein le v els in 3 ′ biotin labeled pre- let-7a-1 pull-down assay. Eluted HA-XPO5 protein was incubated with RAN, GTP and pre- let-7a-1 in the absence or presence of the GST-PIP5K1A. HA-XPO5 and GST-PIP5K1A proteins were detected by western blotting with anti-HA and anti-GST antibodies, respecti v ely. ( D ) Quantification of the enrichment of HA-XPO5. These results were normalized to the input of HA-XPO5 and calculated with ImageJ software. n = 3 independent biological replicates. Error bars r epr esented as mean ± SEM. **** P < 0.0001. ( E, F ) The C / N (Cytoplasmic / Nuclear) ratio of pre- let-7a-1 / pri- let-7a-1 from the indicated cell lines by qRT-PCR analysis. All experiments were performed with at least three biological replicates. All data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001 and NS: not significant. b C t w t t C t X t b c p p t a l l l o l c i f t p n S c u b f P s t v m p O p b m q l i w D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 2024 y an immunofluorescence assay. We found a decreased / N ratio of XPO5 localization in PIP5K1A OE compared o control cells (Supplementary Figure S7A–D) consistent ith the idea that more PIP5K1A could bind with XPO5 o inhibit its function in the export of pre-miRNAs to cyoplasm. Howe v er, we did not observe a difference in the / N ratio of XPO5 localization in PIP5K1A KD compared o control cells (Supplementary Figure S8A–C). Perhaps PO5, after releasing its cargo in the cytoplasm, ra pidl y re- urns to the nucleus to mediate another round of transport. These results indicate that PIP5K1A may function by locking the binding of XPO5 to pre- let-7a-1 . If this conlusion is correct, knockdown of PIP5K1A should increase re- let-7a-1 le v els in the cytoplasmic fraction, because more re- let-7a-1 can bind with XPO5 and then be exported from he nucleus to the cytoplasm. So next we performed a qPCR ssay to test this possibility. We note here that we used preet-7a-1 / pri- let-7a-1 le v els to present the real pre- let-7a-1 e v els as a qPCR assay could not distinguish between preet-7a-1 and pri- let-7a-1 . As expected, we found knockdown f PIP5K1A resulted in higher pre- let-7a-1 / pri- let-7a-1 e v els in cytoplasmic fraction compared to that in control ells (Figure 5 E). We also found PIP5K1A OE resulted n lower pre- let-7a-1 / pri- let-7a-1 le v els in the cytoplasmic raction (Figure 5 F), consistent with the decreased C / N raio of XPO5 localization by immunofluorescent assay (Suplementary Figure S7D). Howe v er, U6 snRNA le v els (a egati v e control) were not changed (Supplementary Figure 9). We confirmed the similar purity of cytoplasmic and nulear fractions under each condition (Supplementary Figre S9). Taken together, these results suggest that PIP5K1A locks the binding of pre- let-7a-1 with XPO5 and then afects the C / N ratio of pre-miRNAs. Ne xt, we inv estigated whether the kinase acti vity of IP5K1A is r equir ed for its function in miRN A bio geneis. First, we created a kinase-dead PIP5K1A mutant proein ( PIP5K1A -D309N), which is already known to preent the production of PIP2 ( 43 ). We confirmed this by imunofluorescence assay with an anti-PIP2 antibody (Sup- lementary Figure S10). We found that PIP5K1A -D309N E cells showed decreased mature let-7a and unchanged re- let-7a-1 le v els relati v e to wild type cells by northern lotting and qRT-PCR assays (Figure 3 H–K), decreased ature let-7b and let-7c le v els relati v e to wild type cells by RT-PCR (Figure 3 K) and lower pre- let-7a-1 / pri- let-7a-1 e v els in the cytoplasmic fraction (Figure 5 F) as also seen n PIP5K1A -WT OE cells. Howe v er, the pri- let-7a-1 le v els ere not changed (Supplementary Figure S11). These re- Figure 6. A schematic model of how PIP5K1A / XPO5 regulates pre-miRNA export from the nucleus to the cytoplasm. D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 2024 sults suggest that the kinase activity of PIP5K1A may be important for pri- let-7 le v els at an early step of miRNA biogenesis, howe v er it is not r equir ed for regulating the export of pre- let-7 from the nucleus and mature let-7 le v els at the later step of miRNA biogenesis. DISCUSSION Taken together, our study points to a previously unrecognized function of PIP5K1A in miRNA biogenesis. In summary, we have found that PIP5K1A regulates the ability of the pre-miRNA / XPO5 complex to participate in miRN A bio genesis / export. We propose a model based on our findings in RKO cell lines (Figure 6 ). In wild type cells, PIP5K1A interferes with the interaction between premiRNAs and XPO5 to maintain normal mature miRNA le v els. Our model explains how PIP5K1A’s interference, which is reduced in PIP5K1A KD cells, allows more premiRNAs to bind to XPO5 and be exported to cytoplasm leading to more mature miRNAs to r epr ess downstr eam gene expression. Our study has demonstrated phosphatidylinositol-4phospha te 5-kinase PPK-1 / PIP5K1A regula tes miRNA le v els from C. elegans to humans. We have found that ppk1 mRN A directl y binds to LIN-28 RN A pr otein fr om our LIN-28 CLIP-seq analysis ( 30 ) and that ppk-1 mRNA levels decrease in a lin-28 ( n719 ) mutant compared to wild type animals by a qRT-PCR assay. Interestingly, we also found that in ppk-1 RNAi animals, lin-28 mRNA le v els decreased compared to control RNAi animals. These results indicate that LIN-28 and PPK-1 function on each other in a feed forward loop. How this occurs will r equir e further analysis. Consistent with lin-28 and ppk-1 acting positi v ely on each other, we found that ppk-1(lf) leads to similar heter ochr onic phenotypes as lin-28(lf). lin-28 mutant animals exhibit a decreased seam cell number as a result of skipping a symmetric cell division step in the L2 stage and exhibit pr ecocious expr ession of adult cell fates. Similar to this, we also found a decreased seam cell number in ppk-1 RNAi animals and a precocious adult cell fate phenotype. Loss of both genes also results in increased let-7 le v els. To answer why ppk-1 RNAi animals showed decreased lin-28 mRNA le v els, we specula te tha t it is possibly because the increase of mature let-7 le v els allows for increased negati v e regulation of its target lin-28 mRNA. It has been reported that the human PIP5K1A mRNA is also bound by LIN28 ( 30 , 44 ) and PIP5K1A positi v ely regulates LIN28 mRNA le v els from our r esults (Figur e 3 M), suggesting that it has a conserved mechanism from C. elegans to humans. We found that PIP5K1A associates with the importin - family factor XPO5 in human cells. While C. elegans PPK-1 is an ortholog of human PIP5K1A, unfortunately C. elegans lacks an XPO5 orthologue ( 45 ). Howe v er C. elegans has XPO-1, which is also an importin -family and seems to function as the major nuclear export receptor ( 45 ). Our preliminary data indicate that PPK-1 associates with XPO1 by a co-immunoprecipitation assay (unpublished data), howe v er this possible interaction needs to be fully investigated in the future. Our results point to further possibilities that warrant future inv estigation. For e xample, PIP5K1A may also hav e roles in other miRN A bio genesis steps, including ( 1 ) possibly transcriptional regulation (altered pri-miRNA levels), and ( 2 ) possibly Drosha / DGCR8 complex-mediated miRNA processing (unchanged pre-miRNA le v els). We have found that pri -let-7a-1 le v els decreased in PIP5K1A KD cells, while its le v els increased in PIP5K1A- WT OE cells, howe v er there was no change in the PIP5K1A -D309N OE cells (Supplementary Figure S11). These results suggest that PIP5K1A positi v ely regulates the pri- let-7a-1 le v els in a kinase dependent manner. As PIP5K1A kinase activity is r o S m s c u n p P c m k b t p a a t g i k t p b c m a T t p ( t fi c t b u w i d v o D T c u S S A W a J c D M D p f A c p f d m F N c C R 1 1 1 1 1 1 1 1 1 D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 2024 eflected in PIP2 le v els (confirmed through ov ere xpression f PIP5K1A -WT OE or PIP5K1A - D309N OE as shown in up Fig. S10), we speculate that PIP5K1A regulates priiRNA le v els through PIP2 le v els. For e xample, PIP2 asociates with histone H1 and RN A pol ymerase II in the nuleus. This possibility should be investigated in the future. We have found that the pre- let-7a-1 le v els did not change pon both depletion or ov ere xpression of PIP5K1A by orthern blotting. Based on the results that PIP5K1A ositi v ely regulates pri- let-7a-1 le v els, we speculate that IP5K1A may negati v ely regulate the Drosha / DGCR8 omplex-mediated miRNA processing to keep normal preiRNA le v els, but how this might happen remains unnown. As LIN28 co-transcriptionally binds pri- let-7 and locks its processing by Drosha / DGCR8 complex ( 35 ), hey may function together in this step. It has been reorted that XPO5 promotes primary miRNA processing in RanGTP-independent manner ( 46 ) and XPO5 also intercts with Dicer mRNA to regulate Dicer expression postranscriptionally ( 47 ). How these additional miRNA bioenesis steps are regulated by PIP5K1A / XPO5 should be nvestigated in the future. A reason for why phospha tid ylinositol 4-phospha te 5- inase participates in miRN A bio genesis is not obvious, alhough it may be a response to signaling during cell cycle rogression or for nuclear membrane integrity ( 48 ). It has een reported that rapid induction of XPO5 occurs during ell cycle entry by a PI3K-dependent post-transcriptional echanism and inhibition of XPO5 results in a prolifertion defect associated with a delayed G1 / S transition. ianyan et al also reported that PIP5K1A interacts with he cell cycle key regulator, CDK1 through formation of rotein-complex es and r egulates cell growth and survival 49 ). The localization of PIP5K1A and its product PIP2 in he nucleus has been reported by other groups and is conrmed by our results. The nuclear envelope (NE) is a physial barrier that regulates nucleocytoplasmic traffic and conrols nuclear e v ents. Since the maintenance of nuclear memrane integrity is essential for normal cell function, we specla te tha t it may make sense to connect nuclear PIP5K1A ith miRN A bio genesis, howe v er this should be addressed n the future. Gi v en that miRNAs are implicated in multiple iseases ( 50 ), including cancer, this new finding might proide a novel therapeutic opportunity to modulate the levels f disease-associated miRNAs. A T A A V AILABILITY he da tasets genera ted during and / or analyzed during the urr ent study ar e available from the corr esponding author pon request. UPPLEMENT ARY DA T A upplementary Data are available at NAR Online. CKNOWLEDGEMENTS e w ould lik e to thank the CGC for C. elegans strains nd Dr Hitoshi Sawa (National Institute of Genetics, apan) and Dr Dipanjan Cho w dhury (Dana-Farber Caner Institute / Harvard Medical School) for plasmids and r Kevin Haigis (Dana-Farber Cancer Institute / Harvard edical School) for cell lines. We thank Dr Manel Esteller, r Christos Miliotis and Tanvi Saxena for advice on ex- eriments and Dr La tika Ma tai and Dr Maria Mavrikaki or comments on the manuscript. We also thank Lay-Hong ng and Aniket Gad of the Confocal Imaging Core Faility at BIDMC for analysis for immunofluorescence exeriments. RNA-seq differential expression testing was perormed by the HSPH Bioinformatics Core. C.L. and F.J.S. esigned the project; C.L., B.Y. and G.S. performed experients and analyzed data; C.L. and F.J.S. wrote the paper. UNDING IA award [R01 AG058816 to F.J.S.]. Funding for open acess charge: NIA [R01 AG058816]. onflict of interest statement. None declared. EFERENCES 1. Lee,R.C., Feinbaum,R.L. and Ambros,V. (1993) The C. elegans heter ochr onic gene lin-4 encodes small rnas with antisense complementarity to lin-14. Cell , 75 , 843–854. 2. Reinhart,B.J ., Slack,F.J ., Basson,M., Pasquinelli,A.E., Bettinger,J .C., Rougvie,A.E., Horvitz,H.R. and Ruvkun,G. (2000) The 21-nucleotide let-7 RNA regulates de v elopmental timing in Caenorhabditis elegans. Nature , 403 , 901–906. 3. Pasquinelli,A.E., Reinhart,B.J., Slack,F., Martindale,M.Q., Kuroda,M.I., Maller,B., Hayward,D.C., Ball,E.E., Degnan,B., Müller,P. et al. (2000) Conservation of the sequence and temporal expression of let-7 heter ochr onic regulatory RNA. Nature , 408 , 86–89. 4. Kim,V.N., Han,J. and Siomi,M.C. (2009) Biogenesis of small rnas in animals. Nat. Rev. Mol. Cell Biol. , 10 , 126–139. 5. Bartel,D.P. (2009) MicroRNAs: target recognition and regulatory functions. Cell , 136 , 215–233. 6. Lund,E., Güttinger,S., Calado,A., Dahlberg,J.E. and Kutay,U. (2004) Nuclear export of MicroRNA precursors. Science , 303 , 95–98. 7. Wu,K., He,J., Pu,W. and Peng,Y. (2018) The role of exportin-5 in MicroRN A bio genesis and cancer. Genomics Proteomics Bioinformatics , 16 , 120–126. 8. Yi,R., Doehle,B.P., Qin,Y., Macara,I.G. and Cullen,B.R. (2005) Ov ere xpression of exportin 5 enhances RNA interference mediated by short hairpin rnas and microRNAs. RNA , 11 , 220–226. 9. Rougvie,A.E. and Moss,E.G. (2013) De v elopmental transitions in C. elegans larval stages. Curr. Top. Dev. Biol. , 105 , 153–180. 0. Wightman,B., Ha,I. and Ruvkun,G. (1993) Posttranscriptional regulation of the heter ochr onic gene lin-14 by lin-4 mediates temporal pa ttern forma tion in C . elegans. Cell , 75 , 855–862. 1. Ambros,V. and Horvitz,H.R. (1984) Heterochronic mutants of the nematode caenorhabditis elegans. Science , 226 , 409–416. 2. Banerjee,D. and Slack,F. (2002) Control of de v elopmental timing by small temporal rnas: a paradigm for RNA-mediated regulation of gene expression. Bioessays , 24 , 119–129. 3. Thornton,J.E. and Gregory,R.I. (2012) How does Lin28 let-7 control de v elopment and disease? T r ends Cell Biol. , 22 , 474–482. 4. Moss,E.G., Lee,R.C. and Ambros,V. (1997) The cold shock domain protein LIN-28 controls de v elopmental timing in C. elegans and is regulated by the lin-4 RNA. Cell , 88 , 637–646. 5. Toker,A. (1998) The synthesis and cellular roles of phospha tid ylinositol 4,5-bisphospha te. Curr. Opin. Cell Biol. , 10 , 254–261. 6. Yu,H., Fukami,K., Watanabe,Y., Ozaki,C. and Takenawa,T. (1998) Phospha tid ylinositol 4,5-bisphospha te re v erses the inhibition of RNA transcription caused by histone H1. Eur. J. Biochem. , 251 , 281–287. 7. Osborne,S.L., Thomas,C.L., Gschmeissner,S. and Schiavo,G. (2001) Nuclear PtdIns(4,5)P2 assembles in a mitotically regulated particle involved in pre-mRNA splicing. J. Cell Sci. , 114 , 2501–2511. 8. Semenas,J., Hedblom,A., Miftakhova,R.R., Sarwar,M., Larsson,R., Shcherbina,L., Johansson,M.E., H ̈ark ̈onen,P., Sterner,O. and D ow nloaded from https://academ ic.oup.com /nar/article/51/18/9849/7257933 by guest on 16 January 2024 Persson,J.L. (2014) The role of PI3K / AKT-related PIP5K1 and the discovery of its selective inhibitor for treatment of advanced prostate cancer. Proc. Natl. Acad. Sci. U.S.A. , 111 , E3689–E3698. 19. Xu,X., Guo,H., Wycuff,D.L. and Lee,M. (2007) Role of phospha tid ylinositol-4-phospha te 5’ kinase (ppk-1) in ovulation of Caenorhabditis elegans . Exp. Cell. Res. , 313 , 2465–2475. 20. Niwa,R., Zhou,F., Li,C. and Slack,F.J. (2008) The expression of the Alzheimer’s amyloid precursor protein-like gene is regulated by de v elopmental timing microRNAs and their targets in Caenorhabditis elegans. Dev. Biol. , 315 , 418–425. 21. Abrahante,J.E., Miller,E.A. and Rougvie,A.E. (1998) Identification of heter ochr onic mutants in Caenorhabditis elegans . Temporal misexpression of a collagen::Green fluorescent protein fusion gene. Genetics , 149 , 1335–1351. 22. Abbott,A.L., Alvarez-Saavedra,E., Miska,E.A., Lau,N.C., Bartel,D.P., Horvitz,H.R. and Ambros,V. (2005) The let-7 MicroRN A famil y members mir -48, mir -84, and mir -241 function together to regulate de v elopmental timing in Caenorhabditis elegans . Dev. Cell , 9 , 403–414. 23. Hada,K., Asahina,M., Hasegawa,H., Kanaho,Y., Slack,F.J. and Niwa,R. (2010) The nuclear receptor gene nhr-25 plays multiple roles in the Caenorhabditis elegans heter ochr onic gene network to control the larva-to-adult transition. Dev. Biol. , 344 , 1100–1109. 24. Chu,Y.D., Wang,W.C., Chen,S.A., Hsu,Y.T., Yeh,M.W., Slack,F.J. and Chan,S.P. (2014) RACK-1 regulates let-7 microRNA expression and terminal cell dif ferentia tion in caenorhabditis elegans. Cell Cy c le , 13 , 1995–2009. 25. Evans,T. (2006) In: T r ansformation and Microinjection . WormBook. 26. Kamath,R. (2003) Genome-wide rnai screening in Caenorhabditis elegans . Methods , 30 , 313–321. 27. Jeon,M., Gardner Heather,F., Miller Eric,A., Deshler,J. and Rougvie Ann,E. (1999) Similarity of the C. elegans de v elopmental timing protein LIN-42 to circadian rhythm proteins. Science , 286 , 1141–1146. 28. Gagnon,K.T., Li,L., Janowski,B.A. and Corey,D.R. (2014) Analysis of nuclear RNA interference in human cells by subcellular fractionation and argonaute loading. Nat. Protoc. , 9 , 2045–2060. 29. Choudhury,S .R., Dutta,S ., Bhaduri,U. and Rao,M.R.S . (2021) LncRNA hmrhl regulates expression of cancer related genes in chronic myelogenous leukemia through chromatin association. NAR Cancer , 3 , zcab042. 30. Stefani,G., Chen,X., Zhao,H. and Slack,F.J. (2015) A novel mechanism of LIN-28 regulation of let-7 microRNA expression re v ealed by in vivo HITS-CLIP in C. elegans. RNA , 21 , 985–996. 31. Rausch,M., Ecsedi,M., Bartake,H., Mullner,A. and Grosshans,H. (2015) A genetic interactome of the let-7 microRNA in C. elegans. Dev. Biol. , 401 , 276–286. 32. Parry,D.H., Xu,J. and Ruvkun,G. (2007) A whole-genome rnai screen for C. elegans miRNA pathway genes. Curr. Biol. , 17 , 2013–2022. 33. van den Bout,I. and Di v echa,N. (2009) PIP5K-dri v en PtdIns(4,5)P2 synthesis: regulation and cellular functions. J. Cell Sci. , 122 , 3837–3850. 34. Weinkove,D., Bastiani,M., Chessa,T.A., Joshi,D., Hauth,L., Cooke,F.T., Di v echa,N. and Schuske,K. (2008) Ov ere xpression of PPK-1, the Caenorhabditis elegans type I PIP kinase, inhibits growth cone collapse in the de v eloping nervous system and causes axonal degeneration in adults. Dev. Biol. , 313 , 384–397. C © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic A This is an Open Access article distributed under the terms of the Creati v e Commo permits unrestricted reuse, distribution, and reproduction in any medium, provided t 35. Van Wynsberghe,P.M., Kai,Z.S., Massirer,K.B., Burton,V.H., Yeo,G.W. and Pasquinelli,A.E. (2011) LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans. Nat. Struct. Mol. Biol. , 18 , 302–308. 36. Rougvie,A.E. and Ambros,V. (1995) The heterochronic gene lin-29 encodes a zinc finger protein that controls a terminal dif ferentia tion e v ent in Caenorhabditis elegans . Development , 121 , 2491–2500. 37. Melo,S.A., Moutinho,C., Ropero,S., Calin,G.A., Rossi,S., Spizzo,R., Fernandez,A.F., Davalos,V., Villanueva,A., Montoya,G. et al. (2010) A genetic defect in exportin-5 traps precursor microRNAs in the nucleus of cancer cells. Cancer Cell , 18 , 303–315. 38. Balz eau,J., Menez es,M.R., Cao,S. and Hagan,J.P. (2017) The LIN28 / let-7 pathway in cancer. Front. Genet. , 8 , 31. 39. Esquela-Kerscher,A. (2014) The lin-4 microRNA: the ultimate micromanager. Cell Cy c le , 13 , 1060–1061. 40. Brownawell,A.M. and Macara,I.G. (2002) Exportin-5, a novel karyopherin, mediates nuclear export of double-stranded RNA binding proteins. J. Cell Biol. , 156 , 53–64. 41. Melo,S.A. and Esteller,M. (2014) Disruption of microRNA nuclear transport in human cancer. Semin. Cancer Biol. , 27 , 46–51. 42. Okada,C., Yamashita,E., Lee,S.J., Shibata,S., Katahira,J., Nakagawa,A., Yoneda,Y. and Tsukihara,T. (2009) A high-resolution structure of the pre-microRNA nuclear export machinery. Science , 326 , 1275–1279. 43. Coppolino,M.G., Dierckman,R., Loijens,J., Collins,R.F., Pouladi,M., Jongstra-Bilen,J., Schreiber,A.D., Trimble,W.S., Anderson,R. and Grinstein,S. (2002) Inhibition of phospha tid ylinositol-4-phospha te 5-kinase ialpha impairs localized actin remodeling and suppresses phagocytosis. J. Biol. Chem. , 277 , 43849–43857. 44. Graf,R., Munschauer,M., Mastrobuoni,G., Mayr,F., Heinemann,U., Kempa,S., Rajewsky,N. and Landthaler,M. (2013) Identification of LIN28B-bound mRNAs re v eals features of target recognition and regulation. RNA Biol. , 10 , 1146–1159. 45. Bussing,I., Y ang,J .S., Lai,E.C. and Grosshans,H. (2010) The nuclear export receptor XPO-1 supports primary miRNA processing in C. elegans and drosophila. EMBO J. , 29 , 1830–1839. 46. Wang,J ., Lee,J .E., Riemond y,K., Yu,Y., Marquez,S.M., Lai,E.C . and Yi,R. (2020) XPO5 promotes primary miRNA processing independently of RanGTP. Nat. Commun. , 11 , 1845. 47. Bennasser,Y., Chable-Bessia,C., Triboulet,R., Gibbings,D., Gwizdek,C ., Dargemont,C ., Kremer,E.J., Voinnet,O. and Benkirane,M. (2011) Competition for XPO5 binding between Dicer mRN A, pre-miRN A and viral RN A regulates human Dicer le v els. Nat. Struct. Mol. Biol. , 18 , 323–327. 48. Iwasaki,Y.W., Kiga,K., Ka y o,H., Fukuda-Yuzawa,Y., Weise,J., Inada,T., Tomita,M., Ishihama,Y. and Fukao,T. (2013) Global microRNA elevation by inducible exportin 5 regulates cell cycle entry. RNA , 19 , 490–497. 49. Wang,T., Sarwar,M., Whitchurch,J.B., Collins,H.M., Green,T., Semenas,J., Ali,A., Roberts,C.J., Morris,R.D., Hubert,M. et al. (2022) PIP5K1 is r equir ed for promoting tumor progression in castra tion-resistant prosta te cancer. Fr ont. Cell Dev. Biol. , 10 , 798590. 50. Slack,F.J. and Chinnaiyan,A.M. (2019) The role of non-coding RNAs in oncology. Cell , 179 , 1033–1055. cids Research. ns Attribution License (http: // creati v ecommons.org / licenses / by / 4.0 / ), which he original work is properly cited.

Article Images (0)

Bioz is the world’s most comprehensive AI search engine for scientific experimentation. The Bioz search engine offers researchers billions of data-driven product, technique, and protocol recommendations. Bioz taps into the latest advances in Artificial Intelligence (AI) – including Natural Language Processing (NLP), Machine Learning (ML) and Generative AI to mine and structure hundreds of millions of pages of complex and unstructured scientific papers. By harnessing the power of AI, Bioz provides researchers with an unprecedented amount of summarized scientific experimentation knowledge right at their fingertips, ultimately helping to speed up drug discovery and the rate of success in finding cures for diseases. Bioz is used by over 15 Million researchers from over 17,000 different universities and companies in 196 countries.